Polychlorinated biphenyls (PCBs) represent a highly problematic environmental pollutant. They are ubiquitous in nature due to their widespread use as electrical insulators, and because of their unreactive chemical structure and low water solubility, they are generally considered to be nondegradable (Kalmaz et al., 1979, Review. Ecol. Model 6:223-251). PCB-contaminated waste is thus a major component of many environmentally-hazardous sites in need of remediation.
There have been several reports of PCB degradation and partial degradation by mixed (Clark et al., 1979, Appl. Environ. Microbiol. 37:680-685; Furukawa et al., 1978, Appl. Environ. Microbiol. 35:223-227; Huntzinger et al., ed., 1974. In: The chemistry of PCBs, p. 1-16, CRC Press, Inc., Boca Raton, Fla.; Kong and Sayler, 1983, Appl. Environ. Microbiol., 46:666-672; Shiaris and Sayler, 1982, Environ. Sci. Technol. 16:367-369) and pure (Ahmed and Focht, 1973, Can. J. Microbiol., 19:45-52; Jeenes et al., 1982, J. Bacteriol. 150:180-187; Mass et al., 1984, Appl. Environ. Microbiol. 47:947-951; Sayler et al., 1977, Microbial Ecology 3:241-255; Yagi and Sudo, 1980, J. Water Pollut. Control Fed. 52:1035-1043) bacterial cultures. Plasmid mediated degradation of mono-chlorobiphenyls has also been reported (Shields et al., 1985, J. of Bacteriol., 163:882-889). Both anaerobic and aerobic microorganisms capable of partially degrading or degrading certain types of PCBs under certain limited conditions have been reported. The reports include a recombinant bacterium capable of growth on a polychlorinated biphenyl as a sole carbon source (McCullar et al., 1994, Applied and Environ. Microbiol. 60:3833-3839). There have also been reports of consortia or mixed cultures of microorganisms which together are able to degrade PCBs (Brenner et al., 1994, Biodegradation, 5:359-377). Also, there have been reports of microorganisms capable of degrading PCBs when a cosubstrate, such as biphenyl or napthalene is added to the media to serve as a primary carbon source (see Brenner et al., 1994, Biodegradation, 5:359-377).
The aforementioned microorganisms are of limited usefulness and are inefficient in bioremediation methods because they either require the addition of a cosubstrate such as biphenyl or naphthalene, to enable the cometabolism of PCBs, or they are only capable of partially degrading PCBs to PCBs having fewer chlorine atoms or to other chlorinated aromatic molecules which cannot be further metabolized by living organisms. Although cosubstrates are potentially available to assist currently known microorganisms in the degradation of PCBs, typical cosubstrates are organic molecules which are themselves environmentally toxic, such as biphenyl and napthalene.
Thus, there is an unmet need for microorganisms that can be used in efficient and non-polluting bioremediation methods for PCB contaminated sites. It is especially desirable to have a bioremediation method of environmental cleanup that does not require the addition of another pollutant or chemical to the environment, and which utilizes readily available microorganisms. The present invention meets these needs.
The invention includes an isolated bacterium, which is capable of utilizing as a sole carbon source a polychlorinated biphenyl, wherein the polychlorinated biphenyl contains at least two chlorine atoms associated therewith.
In one aspect, the polychlorinated biphenyl is 2,3-dichloro-biphenyl.
In another aspect, the polychlorinated biphenyl is 3,4-dichloro-biphenyl.
In yet another aspect, the polychlorinated biphenyl is 2,2xe2x80x2-dichloro-biphenyl.
In another aspect, the polychlorinated biphenyl is 2,4xe2x80x2-dichloro-biphenyl.
In one embodiment, the bacterium is strain SK-1.
In another embodiment, the bacterium is strain SK-2.
In another embodiment, the bacterium is strain SK-3.
In yet another embodiment, the bacterium is strain SK-4.
The invention also includes an isolated bacterium which is capable of utilizing as a sole carbon source a monochloro-biphenyl.
In one aspect, the monochloro-biphenyl is selected from the group consisting of 2-chlorobiphenyl, 3-chlorobiphenyl and 4-chlorobiphenyl.
In another aspect, the bacterium is strain SK-3.
The invention also includes an isolated bacterium which is capable of utilizing as a sole carbon source 4-chlorobenzoate.
Additionally, the invention includes an isolated bacterium which is capable of utilizing both 4-chlorobenzoate and a mono-chlorobiphenyl, individually, as a sole carbon source.
The invention also includes a consortium of microorganisms comprising a bacterium selected from the group consisting of SK-1, SK-2, SK-3 and SK-4.
The invention further includes a recombinant bacterium, which is capable of utilizing as a sole carbon source a polychlorinated biphenyl, wherein the polychlorinated biphenyl contains at least two chlorine atoms associated therewith.
Also included in the invention is a recombinant yeast, which is capable of utilizing as a sole carbon source a polychlorinated biphenyl, wherein the polychlorinated biphenyl contains at least two chlorine atoms associated therewith.
In addition, the invention includes a recombinant bacteriophage, comprising nucleic acid which when expressed in a bacterium infected with the bacteriophage confers on the bacterium the ability to utilize as a sole carbon source a polychlorinated biphenyl, wherein the polychlorinated biphenyl contains at least two chlorine atoms associated therewith.
The invention includes an isolated nucleic acid encoding an enzyme for degrading a polychlorinated biphenyl, wherein the nucleic acid is obtained from an isolated bacterium which is capable of utilizing as a sole carbon source a polychlorinated biphenyl, wherein the polychlorinated biphenyl contains at least two chlorine atoms associated therewith.
The invention also includes a recombinant enzyme for degrading a polychlorinated biphenyl wherein the recombinant enzyme is obtained from an isolated bacterium which is capable of utilizing as a sole carbon source a polychlorinated biphenyl, wherein the polychlorinated biphenyl contains at least two chlorine atoms associated therewith.
In one aspect, the recombinant enzyme is expressed in a bacterium.
In another aspect, the recombinant enzyme is expressed in a yeast.
In yet another aspect, the recombinant enzyme is substantially pure.
The invention also includes a method for the bioremediation of a PCB-contaminated environment. The method comprises the steps of a) adding an isolated bacterium to the PCB-contaminated environment, wherein the bacterium is capable of utilizing as a sole carbon source a polychlorinated biphenyl, wherein the polychlorinated biphenyl contains at least two chlorine atoms associated therewith, and b) incubating the bacterium in the environment for a period of time sufficient to permit degradation of a polychlorinated biphenyl in the environment, thereby bioremediating the environment.
Also included in the invention is a method for enhancing the bioremediation of a PCB-contaminated environment. The method comprises the steps of a) adding an isolated bacterium to the PCB-contaminated environment, wherein the bacterium is capable of utilizing as a sole carbon source a polychlorinated biphenyl, wherein the polychlorinated biphenyl contains at least two chlorine atoms associated therewith, and b) incubating the bacterium in the environment for a period of time sufficient to permit degradation of a polychlorinated biphenyl in the environment, thereby enhancing the bioremediation of the environment.
The invention further includes a method for the bioremediation of a PCB-contaminated environment. The method comprises the steps of a) adding an isolated bacterium to the PCB-contaminated environment, wherein the bacterium is capable of utilizing as a sole carbon source a polychlorinated biphenyl, wherein the polychlorinated biphenyl contains at least two chlorine atoms associated therewith; b) adding a cosubstrate selected from the group consisting of biphenyl and napthalene to the PCB-contaminated environment whereby cometabolism of the cosubstrate and a PCB may occur, and c) incubating the bacterium in the environment for a period of time sufficient to permit degradation of a polychlorinated biphenyl in the environment, thereby bioremediating the environment.
The invention also includes a method for the bioremediation of a PCB-contaminated environment. The method comprises the steps of a) adding a recombinant enzyme for degrading a polychlorinated biphenyl to the PCB-contaminated environment, wherein the recombinant enzyme is obtained from an isolated bacterium which is capable of utilizing as a sole carbon source a polychlorinated biphenyl, wherein the polychlorinated biphenyl contains at least two chlorine atoms associated therewith, and b) incubating the recombinant enzyme in the PCB-contaminated environment for a period of time sufficient to permit degradation of a polychlorinated biphenyl, thereby bioremediating the environment.
Additionally, the invention includes a method for the bioremediation of PCB-contaminated environment. The method comprises the steps of a) adding to the PCB-contaminated environment, a recombinant microorganism selected from the group consisting of a bacterium, a yeast, and a bacteriophage, wherein the recombinant microorganism comprises a nucleic acid encoding an enzyme for degrading a polychlorinated biphenyl, wherein the nucleic acid is obtained from an isolated bacterium which is capable of utilizing as a sole carbon source a polychlorinated biphenyl, wherein the polychlorinated biphenyl contains at least two chlorine atoms associated therewith, and b) incubating the recombinant microorganism in the PCB-contaminated environment for a period of time sufficient to permit degradation of a polychlorinated biphenyl, thereby bioremediating the environment.
The invention also includes a method for the bioremediation of a PCB-contaminated environment. The method comprises the steps of a) adding an isolated bacterium to the PCB-contaminated environment, wherein the bacterium is capable of utilizing as a sole carbon source a polychlorinated biphenyl, wherein the polychlorinated biphenyl contains at least two chlorine atoms associated therewith; b) adding an isolated bacterium to the PCB-contaminated environment, wherein the bacterium is capable of utilizing a chlorobenzoate as a sole carbon source, and c) incubating the bacteria in the environment for a period of time sufficient to permit degradation of a polychlorinated biphenyl and a chlorobenzoate in the environment, thereby bioremediating the environment.